IN VITRO FERTILIZATION

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1 FERTILITY AND STERILITY VOL. 82, NO. 5, NOVEMBER 2004 Copyright 2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. IN VITRO FERTILIZATION Serum antimüllerian hormone/müllerianinhibiting substance appears to be a more discriminatory marker of assisted reproductive technology outcome than follicle-stimulating hormone, inhibin B, or estradiol André Hazout, M.D., a,b Philippe Bouchard, M.D., c David B. Seifer, M.D., d P. Aussage, B.S., e Anne Marie Junca, Ph.D., b and Paul Cohen-Bacrie, Ph.D. b ART Unit Eylau la Muette, Clinique La Muette, Paris, France Received December 30, 2003; revised and accepted March 22, Reprint requests: André Hazout, M.D., 15 Rue Faraday Paris, France (FAX: ; ahazout@hotmail. com). a ART Unit, Hospital University Bichat-C. Bernard, Paris, France. b ART Unit Eylau la Muette, Clinique La Muette, Paris, France. c Endocrinology, Hospital Saint Antoine, AP-HP, Paris, France. d University of Medicine and Dentistry of New Jersey (UMDNJ) Robert Wood Johnson Medical School, New Brunswick, New Jersey. e MDS Pharma Services, Sèvres Cedex, France /04/$30.00 doi: /j.fertnstert Objective: To test the hypothesis that the concentration of early follicular phase serum antimüllerian hormone (AMH) or müllerian-inhibiting substance (MIS) is a useful marker of ovarian response and assisted reproductive technology (ART) outcome. Design: Retrospective analysis of day 3 serum samples drawn before treatment. Setting: Private ART program. Patient(s): One hundred nine consecutive serum samples from women younger than 42 years of age who were undergoing ovulation induction for IVF. Intervention(s): Follicular aspiration for IVF after ovarian stimulation with FSH in a down-regulated cycle using GnRH-a treatment. Main Outcome Measure(s): Correlations between day 3 serum AMH/MIS, E 2, FSH, inhibin B levels, and IVF outcome (i.e., number of retrieved mature oocytes, number and quality of embryos obtained, ongoing clinical pregnancy rates). Multivariate regression analysis on categorical data was performed to describe a predictive model of clinical pregnancy outcome. Result(s): Mean serum AMH/MIS value for clinical pregnancy (n 38) was 2.4 ng/ml, in comparison to 1.1 ng/ml for those who did not become pregnant (n 71). No differences were noted in mean values for day 3 FSH, inhibin B, or E 2 between groups. Multivariate regression analysis demonstrated that day 3 serum AMH/MIS had the greatest independent contribution in predicting pregnancy outcomes. Conclusion(s): These data demonstrate a strong association between day 3 serum AMH/MIS level and IVF outcome in women younger than 42 years of age. Higher AMH/MIS concentrations are associated with a greater number of mature oocytes, a greater number of embryos, and ultimately a higher clinical pregnancy rate. Furthermore, AMH/MIS may offer greater prognostic value than other currently available serum markers of ART outcome. (Fertil Steril 2004;82: by American Society for Reproductive Medicine.) Key Words: Serum AMH/MIS, IVF outcome, prognostic markers In women undergoing treatment for infertility, the predictive value of serum hormone parameters such as FSH, E 2, and inhibin B remains somewhat controversial, and disappointing. Chronological age still appears to be the most reliable predictive factor (1). A serum marker that could be indicative of the pool of nongrowing, FSH-independent follicles before cyclic recruitment might better predict the ovarian response to ovulation induction for ART than current available serum markers. The dimeric glycoprotein antimüllerian hormone (AMH), also referred to as müllerianinhibiting substance (MIS), is a member of the transforming growth factor- (TGF- ) superfamily (2). First described by Jost, AMH/MIS represses the development of müllerian ducts 1323

2 and is produced by immature Sertoli cells in the fetal testis (3, 4). The ovary has also been found to produce low levels of AMH/MIS, synthesized by granulosa cells, only after birth or at the end of fetal life (5). The role of AMH/MIS in the ovary has remained enigmatic because female AMH/MIS null mice had been reported to be fertile (6). However, in this model the ovaries contain less primordial follicles but a greater number of growing follicles. This suggests that AMH/MIS plays an important role in the regulation of early follicular development and in the response to FSH (7). It is now clear that in the ovary, AMH/MIS regulates steroidogenesis and influences folliculogenesis. Antimüllerian hormone/mis is involved in the negative regulation of aromatase and LH receptor genes, thereby increasing androgen production by theca cells. In addition, in postnatal ovarian tissue, AMH/MIS inhibits the initial recruitment of follicles, because AMH/MIS inhibits the stimulating effects of FSH on small and preantral follicle growth (8). Antimüllerian hormone/mis signals through two serine or threonine kinase receptors, type II, required for ligand binding, and type I, for signal transduction (9). Antimüllerian hormone/ MIS appears to use different receptors in different cell types that express different levels of corepressor or coactivators (9). Experiments in rodent demonstrated that AMH/MIS type II receptors are expressed in granulosa cells (10), suggesting a paracrine action to regulate granulosa cells and oocyte function. In women, the highest values of AMH/MIS are attained after puberty (11), and AMH/MIS is detected in adult human serum. Antimüllerian hormone/mis is produced by granulosa cells of follicles as they start to grow, that is, by preantral and early antral follicle (12), and its secretion ends when a follicle is selected for ovulation. Recent clinical reports demonstrated that early follicular serum AMH/MIS levels are strongly associated with ovarian follicular status (13) and with the number of oocytes retrieved after controlled ovarian stimulation for ART cycles (14). The aim of this study was to test the hypothesis that day 3 serum AMH/MIS may be a more useful prognostic marker of clinical pregnancy in comparison with the currently available markers, which include FSH, E 2, and inhibin B. MATERIALS AND METHODS Samples were analyzed from each of 109 consecutive patients between September 2002 and December 2002 who had serum drawn on day 3 of nontreated cycles, within 3 months before ovarian stimulation for IVF. The samples had been stored at 20 C to 80 C. Women with polycystic ovary syndrome, or who were older than 42 years of age, or who had increased FSH levels ( 12 IU/L) were excluded. We also excluded women with a small number of antral follicles on day 3 (less than five) to obtain a homogeneous group to study. The distribution was as following: 2 women with endometriosis, 49 with male infertility, 33 with oligo-ovulation, 10 with tubal infertility, and 15 with mixed infertility. Pituitary desensitization with daily triptorelin treatment (Decapeptyl, 0.1 mg; Beaufour Ipsen Laboratories, Paris, France) was started in the previous luteal phase. Hormonal control was performed on day 2 of the following cycle before stimulation with daily administration of recombinant FSH (Gonal F, 225 IU; Serono Laboratories, Boulogne, France) until triggering of ovulation with 10,000 IU of hcg IM when more than three follicles with mean diameter of 16 mm were noted. Occasionally, recombinant FSH was replaced by hmg, if serum LH on day 6 of stimulation was 1 IU/L. Follicles measuring 16 mm in diameter were aspirated under patient sedation, 36 hours after hcg injection. This study received institutional review board approval from the ethical committee of our university before it was executed. Each patient gave informed consent authorizing the examination. Antimüllerian Hormone or MIS Serum AMH/MIS levels were determined by using an ultrasensitive ELISA (Beckman-Coulter, Villepinte, France) as described elsewhere (5). For AMH/MIS, functional sensitivity was 0.04 ng/ml; intra-assay and interassay coefficients of variation were 5% and 8%, respectively. The AMH/MIS ELISA does not recognize LH, FSH, activin, inhibin, or TGF- and did not cross-react with bovine or rodent AMH/MIS. Other Hormone Measurements Serum inhibin B levels were determined by using a doubleantibody ELISA (Serotec, Varilhes, France). For inhibin B, functional sensitivity was 15 pg/ml, and intra-assay and interassay coefficients of variation were 6 and 9%, respectively. Serum levels of E 2, FSH, and LH were determined with an automated multi-analysis system with chemiluminescence detection (ACS-180; Bayer Diagnostics, Puteaux, France). For E 2, functional sensitivity was 15 pg/ml, and intraassay and interassay coefficients of variation were 8% and 9%, respectively. For FSH and LH, functional sensitivity was 0.1 miu/ml, and intra-assay and interassay coefficients of variation were 3% and 5%, respectively. Statistical Analysis Descriptive analysis was performed to compare serum levels of each hormone with the number of mature oocytes and to compare the number of embryos with IVF outcome Hazout et al. Serum AMH/MIS: prognostic marker for ART Vol. 82, No. 5, November 2004

3 TABLE 1 Comparison between pregnant and nonpregnant women. Variable Group Age (y) AMH/MIS (ng/ml) FSH (IU/L) Inhibin B (pg/ml) LH (IU/L) E 2 (pg/ml) Nonpregnant group (n 71) Mean SD th percentile th percentile Pregnant group (n 38) Mean * SD th percentile th percentile * P.0017 (nonpregnant group vs. pregnant group). Because of the selection of subjects (screening before IVF), the required normality of data was not achieved to apply the standard multivariate regression approach (logistic or discriminant analysis). Consequently, Optimal Scaling was used to discount this issue and predict a response variable (clinical pregnancy as defined as cardiac activity by transvaginal ultrasound) from a set of predictor variables. This procedure was developed by the Data Theory Scaling System Group (DTSS) Faculty of Social and Behavioral Science, University of Leiden, The Netherlands. The advantage of this approach is that it confirms different predictor cutoff levels and analyzes nonlinear relationships between variables. All predictors were treated as ordinal variables, and success or failure of IVF was treated as a nominal variable. The values of the standardized coefficients reflect the amount of change in the predictive response to IVF. The R-squared values of partial correlations provide the percentage of variance explained by each variable to the model. The importance values describe the relative importance of predictors and aid in interpreting predictor contributions to the regression. Any value that is large relative to the others is important as a predictive variable. The different levels at which each variable value could be scaled, as illustrated in plots called transformation plots, are named quantifications. Transformation plots illustrate intuitively the relationship between the original values of any variable and the quantification of that variable. RESULTS A comparison of mean ages, day 3 serum FSH, AMH/ MIS, inhibin B, LH, number of oocytes, and embryos is summarized in Tables 1 and 2. Mean age, FSH, E 2, inhibin B, and LH were similar among nonpregnant and pregnant women, in contrast to the case of mean day 3 serum AMH/MIS ( ng/ml vs ng/ml; P.002). In Table 2, Spearman correlations reveal that AMH/MIS levels were positively correlated with the number of metaphase II oocytes (r 0.38) and with the total number of embryos (r 0.34). Antimüllerian hormone/mis levels were lower among women who had a shorter preceding cycle duration ( 26 days). To summarize the model fit: regression with optimal scaling yielded an R 2 of 0.42, indicating that 42% of the variance of the transformation outcome (success or failure of clinical pregnancy) was explained by the regression on the optimal transformation predictors. The F statistic of 9.09 with corresponding P values.001 also indicated the strength of the prognostic model. Analysis of the standardized coefficients (Table 3) demonstrated that the largest coefficient occurs for AMH/ MIS ( 0.48). All the other predictors but one (inhibin B, with a poor F value of 0.50) appear to predict the success or the failure of IVF: duration of the preceeding cycle ( 0.37), LH (with a value of 0.28), then age. Folliclestimulating hormone, LH, IVF rank order, and E 2 with absolute values had F ranges from 0.16 to A positive value indicated that failure of IVF was more frequently observed for the lowest values, and success, for the highest values. A negative value indicated that success of IVF was associated with young patient age and that failure was associated with older patient age. Removing the effect of all other variables, AMH/MIS explains 26% of the variance for success or failure with IVF, the duration of the FERTILITY & STERILITY 1325

4 TABLE 2 Spearman correlations. AMH/MIS FSH Inhibin B LH No. of MII oocytes No. of embryos E 2 IVF rank order Duration of previous cycle AMH/MIS 1 FSH Inhibin B LH No. of MII oocytes 0.38 a No. of embryos 0.34 a E IVF rank order Duration of previous cycle 0.33 a Age Note: MII metaphase II. a Statistically significant positive correlations. preceding cycle explains 18% of the variance, and LH explains 10%. Importance of AMH/MIS is thus high (0.41), and this shows that this marker is a crucial predictor. Inhibin B values appear to have no influence on the variation of success or failure to IVF. The first transformation plot describes the quantification of success or failure to IVF. At the bottom of the plot 71 women who failed to IVF have a score of At the top of the graph, the scale value for success to IVF is 1.37 (38 women in this group; Fig. 1). Figure 2 illustrates category quantifications for AMH/ MIS. In this plot, consecutive categories correspond to similar quantifications; such categories result in a plateau, and distinction may be unnecessary. Some of the categories could be combined. Values over the zero quantification, that is, AMH/MIS values 1.1, contribute to the success of IVF. Conversely, AMH/MIS values 1.1 were associated with failure, especially at the 0.2 level. If the duration of the preceeding cycle was 26 days, failure resulting from IVF was more frequently observed; also, more failures were observed after four IVF attempts. According to other studies (15), more failures resulting from IVF (n 12) are observed if FSH values are 10 IU/L. The cutoff in this study was 9.8 IU/L, above which more stimulation attempts could be canceled. Conversely, the ab- FIGURE 1 Category quantification for pregnancy. TABLE 3 Standardized coefficients of regression. Variable Standardized SE F Percentage variance explained Importance AMH/MIS Preceeding cycle length IVF rank order FSH LH Age E Inhibin B Quantifications for "pregnancy" ess No n=38 n=71 Pregnancy Yes -1.0 Success Failure 1326 Hazout et al. Serum AMH/MIS: prognostic marker for ART Vol. 82, No. 5, November 2004

5 FIGURE 2 Category quantification for AMH/MIS. Std Coef. = Importance = 0.40 % explained = 26% 4 Quantification of AMH/MIS N=32 N=37 N=20 N=6 N=11-2 N=2-3 O O Standardized Coefficient: Antimüllerian hormone/ Müllerian inhibiting substance N=1 Success Failure solute value of E 2 was not a determining factor in predicting IVF outcome. There was no clear indication of IVF success or failure between LH levels of 3.80 and 5.70 IU/L, but according to our data, levels 5.70 IU/L were more often associated with failure, and LH levels 3.80 IU/L were more often associated with success. Among the usual markers already suggested by other authors, age is the best predictive factor of success, and in our study, more pregnancies were observed in women aged younger than 33 years. Because women had been screened before their inclusion in our IVF program (FSH 12 IU/L, E 2 50 pg/ml, inhibin B 40 pg/ml), inhibin B values only defined the remaining failures (n 27) when they were 64 pg/ml. This marker was not directly indicative of success in IVF (Fig. 3). DISCUSSION Previous studies have examined the value of AMH/MIS and other markers, using as endpoints oocyte number (14, 16) or follicular count (13). Van Rooij et al. (16) examined ongoing pregnancy in a subset of his patients as a secondary endpoint but did not find a significant association with any serum marker including FSH, E 2, or inhibin B. All these studies concluded that AMH/MIS may be a more useful serum marker than any of the known serum markers for assessment of ovarian reserve. It appears that AMH/MIS has the advantage of reflecting most accurately the pool of primordial and nongrowing FSH-independent follicles before cyclic recruitment. De Vet and al. (17) have also shown that serum AMH/MIS on day 3 decreases with age and becomes undetectable after menopause, supporting the view that AMH/MIS is a remarkable marker of ovarian aging. This has been confirmed in women treated by chemotherapy (18). Our data demonstrate that serum day 3 AMH/MIS level is of great value for predicting clinical pregnancy outcome of IVF. Indeed, serum AMH/MIS measurement had greater prognostic value than age, serum FSH, inhibin B, or E 2. This study is the first to demonstrate the clinical utility of AMH/ MIS in predicting clinical pregnancy as an endpoint. From this last point of view, age, serum day 3 FSH, inhibin B, and E 2 are not significantly different from the nonpregnant women group. FERTILITY & STERILITY 1327

6 FIGURE 3 Category quantification for inhibin B. Quantifications for inhibin B N=82 Failure -1.5 N= Inhibin B Conversely, higher serum AMH/MIS concentrations were associated with significantly higher pregnancy rates. This is of strong interest if we consider that all the women in our IVF program were recruited with an adequate hormonal and sonographic ovarian status. Moreover AMH/MIS is correlated with not only the number of oocytes and embryos but also the length of previous cycles; this appears logical because shortening of the cycle may be one of the first signs of diminished ovarian function. The predictive power of serum AMH/MIS is strong, with 26% of explained variance. The addition of all the other markers is barely enough to reach this significance. When the serum day 3 AMH/MIS value was 1.1, we found that young women were more frequently pregnant than the oldest, except when AMH/MIS level was 0.2 ng/ml. Serum day 3 FSH is commonly referred to as the best predictive factor in ART. Recent studies (1) showed that FSH is not really correlated with ART outcome but only with ovarian response. We confirm here that FSH has no effect on AMH/MIS secretion and confirm the cutoff of 9.8 IU/mL, already defined by other investigators (15), above which there are more canceled cycles. Inhibin B, also secreted by small antral follicles, is definitively only one of the markers of granulosa cell competence and ovarian follicular reserve. This study demonstrated the absence of correlation between day 3 serum AMH/MIS and inhibin B levels and the low predictive power of serum inhibin B in ART outcome. With further study, some combination of one or more of these serum markers in combination with AMH/ MIS may offer greater prognostic value than any single marker alone. Another apparent advantage of AMH/MIS over its alternatives (FSH, inhibin B, E 2 ) may be that its concentration is relatively stable throughout the menstrual cycle (19). We chose to measure AMH/MIS on samples drawn on day 3 only to compare AMH/MIS with the usual markers of the ovarian reserve that are measured on day 3. Thus, unlike other markers, its utility may not be restricted exclusively to an early follicular phase window of sampling before GnRH down-regulation and stimulation. In conclusion, the present results provide the first simultaneous examination of the known prognostic serum markers, using clinical pregnancy as an endpoint. Serum AMH/MIS appears to be the most reliable factor of success. Its relative stability during the cycle, technical ease of examination, and predictive power make it currently, with perhaps sonography, the most discriminatory prognostic marker of outcome in ART. However, further studies are needed to elucidate the physiological role of AMH/ MIS in women. References 1. Chuang CC, Chen CD, Chao KH, Chen SU, Ho HS, Yang YS. Age is a better predictor of pregnancy potential than basal FSH levels in women undergoing IVF. Fertil Steril 2003;79: Pepinski RB, Sinclair LK, Chow EP, Mattaliano RJ, Manganaro TF, Donahoe PK, et al. Proteolytic processing of Müllerian inhibiting substance produces a transforming growth factor like fragment. J Biol Chem 1988;263: Josso N, Legeai L, Forest MG, Chaussin JL, Brauner R. An enzyme linked immunoassay for anti müllerian hormone: a new tool for the evaluation of testicular function in infants and children. J Clin Endocrinol Metab 1990;70: Lee MM, Donahoe PK, Hasegawa T, Silverman B, Crist GB, Best S, et al. Müllerian inhibiting substance in humans: normal levels from infancy to adulthood. J Clin Endocrinol Metab 1996;81: Long WQ, Ranchin V, Pautier P, Belville C, Denizot P, Cailla H, et al. 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Developmental expression of a candidate Müllerian inhibiting substance type II receptor. Endocrinology 1996;137: Hudson PL, Dougla I, Donahoe PK, Cate RL, Epstein J, Pepinsky RB, et al. An immunoassay to detect human Müllerian inhibiting substance in males and females during normal development. J Clin Endocrinol Metab 1990;70: Vigier B, Picard JY, Tran D, Legeai L, Josso N. Production of anti Müllerian hormone: another homology between Sertoli and granulosa cells. Endocrinology 1984;114: Fanchin R, Schonauer LM, Righini C, Guibourdenche J, Frydman R, Taieb J. Serum anti-mullerian hormone is more strongly related to ovarian follicular status than serum inhibin B, estradiol, FSH and LH on day 3. Hum Reprod 2003;18; Hazout et al. Serum AMH/MIS: prognostic marker for ART Vol. 82, No. 5, November 2004

7 14. Seifer DB, MacLaughlin DT, Christian BP, Feng B, Shelden RM. Early follicular serum mullerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertil Steril 2002;77: Creus M, Peñarrubia J, Fábregues F, Vidal E, Carmona F, Casamitjana R, et al. Day 3 serum inhibin B and FSH and age as predictors of assisted reproduction treatment outcome. Hum Reprod 2000;15: Van Rooij IAJ, Broekmans FJM, te Velde ER, Fauser BCJM, Bancsi LFJMM, de Jong FH, et al. Serum anti-mullerian hormone levels: a novel measure of ovarian reserve. Hum Reprod 2002;17: De Vet A, Laven JS, de Jong FH, Themmen AP, Fauser BC. Anti Müllerian hormone serum levels: a putative marker for ovarian aging. Fertil Steril 2002;77: Bath LE, Wallace WHB, Shaw MP, Fitzpatrick C, Anderson RA. Depletion of ovarian reserve in young women after treatment for cancer in childhood: detection by anti Müllerian hormone, inhibin B and ovarian ultrasound. Hum Reprod 2003;18: Cook CL, Siow Y, Taylor S, Fallat ME. Serum mullerian-inhibiting substance levels during normal menstrual cycles. Fertil Steril 2000;73: FERTILITY & STERILITY 1329